J. Bras. Patol. Med. Lab. vol.40 número1

Infectivity฀of฀wild-type฀rubella฀virus฀฀

in฀fibrochondrocyte฀cells฀฀

Infectividade฀do฀vírus฀selvagem฀da฀rubéola฀em฀células฀de฀fibrocondrócitos

Cristina฀A.฀Figueiredo;฀Maria฀I.฀Oliveira;฀Ana฀M.฀S.฀Afonso;฀Suely฀P.฀Curti J฀Bras฀Patol฀Med฀Lab฀•฀v.฀40฀•฀n.฀1฀•฀p.฀1-5฀•฀fevereiro฀2004

Serviço฀de฀Virologia/Divisão฀de฀Biologia฀Médica฀do฀Instituto฀Adolfo฀Lutz฀(SP).

abstract

This฀study฀describes฀the฀rapid฀growth฀of฀the฀rubella฀virus฀in฀samples฀of฀a฀primary฀fibrochondrocyte฀cell฀ culture฀with฀the฀development฀of฀a฀cytopathic฀effect฀(CPE),฀in฀response฀to฀infection฀by฀the฀rubella฀virus.฀The฀ cells฀were฀isolated฀from฀the฀meniscus฀joint฀of฀a฀rabbit฀after฀enzymatic฀extraction฀and฀incubated฀at฀37°C฀ with฀a฀Dulbecco’s฀modified฀Eagle’s฀medium฀(DMEM),฀supplemented฀with฀10%฀fetal฀calf฀serum.฀A฀total฀ of฀six฀clinical฀samples฀from฀urine,฀blood฀and฀cerebrospinal฀fluid฀were฀inoculated฀in฀the฀fibrochondrocyte฀ and฀the฀cell฀lines฀of฀the฀African฀green฀monkey฀kidney฀–฀ATCC฀CCL-81฀(Vero).฀The฀fibrochondrocyte฀cell฀ showed฀CPE฀after฀24฀hours฀and฀virus฀growth฀was฀confirmed฀by฀immunohistochemistry฀and฀ Nested฀PCR.฀The฀cells฀inoculated฀with฀samples฀were฀examined฀by฀phase฀contrast฀microscopy฀ and฀showed฀characteristic฀rounding,฀along฀with฀bipolar฀and฀multipolar฀cells.฀The฀infection฀ curve฀increased฀during฀the฀five฀days฀of฀observation,฀showing฀that฀the฀titers฀in฀fibrochondrocyte฀ cells฀were฀then฀higher฀than฀those฀observed฀in฀Vero฀cell฀lines.

Fibrochondrocytes

Rubella฀virus

Isolation

resumo

Este฀trabalho฀descreve฀o฀rápido฀crescimento฀do฀vírus฀da฀rubéola฀em฀amostras฀clínicas฀em฀cultura฀primária฀de฀ fibrocondrócitos฀com฀desenvolvimento฀de฀efeito฀citopático฀em฀resposta฀a฀infecção฀pelo฀vírus฀da฀rubéola.฀As฀células฀ foram฀isoladas฀do฀menisco฀do฀joelho฀do฀coelho฀após฀extração฀enzimática฀e฀incubadas฀a฀37°C฀em฀meio฀DMEM฀ suplementado฀com฀10%฀de฀soro฀fetal฀bovino.฀Seis฀amostras฀clínicas฀de฀urina,฀sangue฀e฀liquor฀foram฀inoculadas฀ na฀cultura฀primária฀de฀fibrocondrócitos฀e฀na฀linhagem฀Vero.฀As฀células฀de฀fibrocondrócitos฀apresentaram฀efeito฀ citopático฀após฀24฀horas฀de฀incubação฀e฀o฀vírus฀foi฀detectado฀por฀imunoistoquímica฀e฀Nested฀PCR.฀As฀células฀ infectadas฀apresentaram฀aspecto฀arredondado,฀com฀formação฀de฀alguns฀prolongamentos฀citoplasmáticos฀e฀ sincícios,฀produzindo฀células฀multinucleadas.฀A฀curva฀de฀crescimento฀da฀infecciosidade฀do฀vírus฀foi฀mais฀alta฀ quando฀comparada฀com฀a฀linhagem฀celular฀Vero.

Fibrocondrócitos

Vírus฀da฀rubéola

Isolamento

unitermos

y

key words

0RIGINAL฀PAPER

Introduction

The฀menisci฀of฀the฀knee฀are฀semilunar,฀fibrocartilaginous฀ elements฀ situated฀ between฀ the฀ femoral฀ condyles฀ and฀ tibial฀ plateaus.฀ The฀ fibrocartilage฀ consists฀ of฀ cells฀ (fibrochondrocytes฀)฀surrounded฀by฀an฀abundant฀extracellular฀ matrix(14,฀18,฀19)_{.฀Two฀populations฀of฀fibrochondrocytes฀can฀}

be฀discerned฀at฀both฀the฀light฀and฀electron฀microscopic฀ levels,฀both฀resembling฀typical฀chondrocytes(9,฀ 10)_{.฀Unlike฀}

fibroblasts,฀the฀fibrochondrocytes฀are฀either฀round฀or฀oval,฀ and฀are฀situated฀in฀lacunae(11)_.

Rheumatoid฀arthritis฀(RA)฀is฀the฀most฀common฀type฀of฀ inflammatory฀arthritis,฀affecting฀about฀1%฀of฀the฀general฀ population฀worldwide.฀The฀etiology฀of฀RA฀remains฀a฀mystery,฀ but฀a฀variety฀of฀studies฀suggest฀that฀a฀blend฀of฀environmental฀ and฀genetic฀factors฀is฀responsible.฀The฀contribution฀of฀either฀ one฀is฀necessary฀but฀not฀sufficient฀for฀full฀expression฀of฀the฀ disease.฀Extensive฀research฀in฀this฀regard฀has฀been฀negative.฀ Similarly,฀considerable฀attention฀has฀been฀directed฀at฀the฀ potential฀role฀for฀viruses฀in฀arthritis฀and฀the฀possible฀cause฀ of฀infection,฀including฀the฀rubella฀virus(3,฀7,฀8)_.

Rubella฀ virus฀ is฀ a฀ RNA฀ virus฀ which,฀ most฀ commonly,฀ only฀causes฀an฀acute,฀mild฀systemic฀illness฀characterized฀by฀ fever฀and฀exanthema(2)_{.฀However,฀the฀association฀of฀rubella฀}

virus฀with฀acute,฀transient฀joint฀manifestations,฀after฀both฀ natural฀infection฀and฀vaccination,฀has฀been฀recognized฀for฀ many฀years(4,฀16,฀17)_.

The฀ present฀ study฀ showed฀ the฀ rapid฀ growth฀ of฀ the฀ rubella฀virus฀in฀fibrochondrocyte฀cells฀isolated฀from฀rabbit฀ knee-joints฀with฀the฀development฀of฀cytopathic฀effects฀in฀ response฀ to฀ infection฀ by฀ rubella฀ virus.฀ The฀ rubella฀ virus฀ isolated฀from฀the฀samples฀were฀titered฀simultaneously฀in฀ the฀African฀green฀monkey฀kidney฀–฀ATCC฀CCL-81฀(Vero)฀ cells฀and฀fibrochondrocyte฀cells.

Materials and methods

Rubella฀virus฀positive฀samples

The฀six฀positive฀rubella฀virus฀clinical฀samples฀(of฀blood,฀ urine฀ and฀ cerebrospinal฀ fluid)฀ used฀ in฀ this฀ study฀ were฀ obtained฀during฀screening฀for฀rubella฀or฀measles฀virus.฀Two฀ samples฀of฀the฀peripheral฀blood฀lymphocytes฀were฀separated฀ from฀heparinized฀blood฀with฀ficoll-hypaque฀gradients฀and฀ suspended฀in฀Dulbecco’s฀modified฀Eagle’s฀medium฀(DMEM฀ –฀Gibco,฀Grand฀Island,฀NY,฀USA)฀supplemented฀by฀2%฀fetal฀ calf฀serum฀(FCS).฀The฀samples฀were฀stored฀at฀-70°C฀until฀ further฀process.

Primary฀cell฀culture

The฀knee฀joints฀of฀male฀and฀female฀New฀Zealand฀white฀ rabbits฀ of฀ six฀ months฀ of฀ age฀ were฀ exposed฀ to฀ aseptic฀ conditions.฀The฀lateral฀and฀medial฀menisci฀were฀surgically฀ removed฀ from฀ knee-joints฀ and฀ rinsed฀ in฀ saline฀ solution,฀ pH฀ 7.5,฀ containing฀ 40µg/mL฀ of฀ gentamicin฀ (Schering,฀ Corp.).Then,฀ the฀ menisci฀ were฀ placed฀ in฀ Petri฀ dishes฀ containing฀DMEM฀with฀10%฀FCS,฀2mM฀L-glutamine฀and฀ 40µg/mL฀ gentamicin฀ (Schering,฀ Corp.)฀ The฀ cells฀ were฀ isolated฀by฀sequential฀treatment฀of฀the฀minced฀tissue฀with฀ 2mg/mL฀clostridial฀collagenase฀(Gibco,฀Grand฀Island,฀NY,฀ USA)฀ in฀ DMEM฀ with฀ 10%฀ FCS,฀ 2mM฀ L-glutamine฀ and฀ 40µg/mL฀gentamicin฀(Schering,฀Corp.)฀to฀release฀the฀cells฀ from฀the฀tissue.฀The฀cells฀were฀then฀centrifugated฀at฀250x฀ g฀for฀ten฀minutes฀and฀washed฀with฀DMEM฀containing฀10%฀ FCS.฀Cell฀number฀was฀determined฀using฀a฀hematocytometric฀ chamber,฀and฀cell฀viability฀was฀assessed฀by฀trypan฀blue฀(0.1%)฀ exclusion.฀Primary฀cultures฀were฀started฀by฀inoculating฀5x105_฀

cells/mL฀in฀T25฀plastic฀culture฀flasks฀containing฀10mL฀of฀ DMEM฀supplemented฀with฀10%฀FCS฀and฀were฀maintained฀ at฀37°C.฀The฀cells฀were฀submitted฀to฀incubation฀at฀37°C฀in฀a฀ water-saturated฀atmosphere฀with฀5%฀CO₂.฀The฀first฀passage฀ was฀established฀from฀cells฀obtained฀by฀trypsin฀treatment฀of฀ confluent฀primary฀cultures฀and฀fibrochondrocytes฀were฀split฀ 1:2฀with฀DMEM฀supplemented฀as฀described฀above.

Primary฀ and฀ first฀ passage฀ cultures฀ were฀ used฀ in฀ this฀ study฀because฀of฀the฀cells฀retaining฀many฀of฀their฀in฀vivo฀ phenotypic฀characteristics.

Virus฀growth

The฀cell฀line฀of฀the฀African฀green฀monkey฀kidney฀–฀ATCC฀ CCL-81฀(Vero)฀and฀fibrochondrocyte฀cells฀containing฀1฀x฀106_฀

cells/mL฀were฀grown฀in฀T25฀flasks฀in฀DMEM฀supplemented฀ with฀ 10%฀ FCS,฀ 20mM฀ L-glutamine.฀ The฀ confluent฀ cells฀ were฀inoculated฀with฀0.5mL฀RA฀27/3฀virus฀strain฀(Meruvax฀ II,฀Merck,฀Sharp฀and฀Dohme)฀and฀0.5mL฀of฀each฀sample฀ for฀ one฀ hour฀ at฀ room฀ temperature.฀ After฀ one฀ hour฀ of฀ adsorption,฀each฀cell฀culture฀received฀5mL฀of฀medium฀with฀ 2%฀FCS฀and฀were฀incubated฀at฀37°C.฀The฀medium฀was฀ replaced฀every฀three฀days.฀Cell฀cultures฀were฀observed฀for฀ CPE฀daily฀during฀seven฀days.฀After฀this฀time฀period,฀the฀cells฀ were฀harvested,฀frozen,฀and฀thawed฀once,฀and฀the฀clarified฀ supernatant฀ was฀ stored฀ in฀ aliquots฀ at฀ -70°C.฀ Uninfected฀ cultures฀were฀also฀prepared฀and฀treated฀identically.

Virus฀stock

in฀Vero฀and฀fibrochondrocyte฀cells.฀The฀cells฀containing฀2฀ x฀106_{฀cells฀were฀grown฀in฀T25฀flasks฀in฀DMEM฀with฀10%฀}

FCS฀and฀infected฀with฀0.5mL฀of฀the฀RA฀27/3฀virus฀strain฀ and฀ positive฀ rubella฀ samples.฀ The฀ cells฀ were฀ maintained฀ in฀DMEM฀supplemented฀with฀2%฀fetal฀calf฀serum,฀20mM฀฀ L-glutamine฀and฀0.1%฀gentamicin.฀For฀the฀virus฀stock,฀three฀ passages฀were฀performed฀on฀cell฀cultures.

Titration฀of฀virus

The฀first฀passage฀Vero฀cell฀line฀and฀fibrochondrocyte฀cells฀ (1฀x฀105_{฀cells/mL)฀were฀grown฀in฀24฀well฀plates฀in฀DMEM,฀}

supplemented฀with฀2mM฀L-glutamine฀and฀10%฀FCS.฀Plates฀ were฀incubated฀at฀37°C฀in฀a฀humidified฀5%฀CO₂฀atmosphere.฀ A฀serial฀tenfold฀dilution฀of฀the฀rubella฀virus฀(RA฀27/3฀strain฀ and฀positive฀rubella฀samples)฀were฀prepared฀in฀DMEM.฀The฀ confluent฀ cells฀ were฀ inoculated฀ with฀ 200µL฀ of฀ the฀ virus฀ dilutions฀in฀quadruplicates,฀for฀each฀different฀cell฀lines.฀After฀ one฀ hour฀ of฀ adsorption฀ at฀ room฀ temperature,฀ each฀ well฀ received฀2mL฀of฀medium฀DMEM฀with฀2%฀FCS.฀Uninfected฀ cultures฀were฀also฀prepared฀and฀treated฀identically฀as฀controls.฀ Plate฀cultures฀were฀observed฀for฀CPE฀daily฀during฀seven฀days,฀ when฀the฀test฀was฀concluded.฀Fifty฀per฀cent฀infectivity฀end฀ points฀were฀calculated฀by฀the฀method฀of฀Karber(12)_{.฀All฀titers฀}

are฀given฀as฀log฀10฀TCID₅₀฀per฀.0.1mL฀of฀virus.

Optic฀microscopy

The฀infected฀and฀uninfected฀fibrochondrocyte฀cells฀were฀ cultivated฀ on฀ coverslips฀ for฀ staining฀ with฀ blue฀ toluidine฀ and฀hematoxylin-eosin฀(H&E).The฀cells฀were฀rinsed฀with฀ cacodylate฀buffer฀and฀sucrose฀0.2M฀for฀30฀min฀at฀room฀ temperature.฀ The฀ cultures฀ were฀ fixed฀in฀ situ฀ with฀ 1%฀ glutaraldehyde฀in฀0.15M฀phosphate฀buffer฀at฀pH฀7.2฀for฀one฀ hour฀at฀4°C.฀After฀being฀rinsed฀twice฀with฀cacodylate฀buffer฀ and฀stained฀directly฀with฀toluidine฀blue฀and฀H&E฀the฀cells฀ were฀examined฀by฀light฀microscopy฀and฀photographed.

The฀blue฀toluidine฀specifies฀fibrocartilage฀and฀H&E฀stain฀ makes฀CPE฀evident.

Immunoperoxidase฀staining฀of฀RV฀antigen

Fibrochondrocyte฀cells฀in฀first฀passage฀were฀grown฀in฀ micro฀tissue฀culture฀chamber฀slides฀(Nunc฀Inc.,฀Naperville,฀ IL฀)฀at฀a฀concentration฀of฀1฀x฀105_{฀cells/mL฀and฀incubated฀at฀}

37°C฀in฀a฀humidified฀5%฀CO₂฀atmosphere,฀then฀the฀cultures฀ confluent฀monolayers฀were฀infected฀with฀0.01฀moi฀rubella฀ virus฀isolated฀from฀urine฀and฀RA฀27/3฀strain.฀After฀one฀hour฀ of฀adsorption฀at฀room฀temperature,฀1mL฀of฀maintenance฀ medium฀ was฀ added.฀ Uninfected฀ cells฀ were฀ included฀ as฀ negative฀controls.

After฀ three฀ days,฀ the฀ cell฀ monolayers฀ were฀ rinsed฀ with฀ phosphate-buffered฀ saline฀ (PBS)฀ and฀ fixed฀ with฀ 4%฀ paraformaldehyde฀ in฀ phosphate฀ buffer.฀ The฀ cells฀ were฀ then฀ treated฀ with฀ 1.8%฀ hydrogen฀ peroxide฀ in฀ methanol฀ to฀ remove฀ endogenous฀ peroxidase฀ activity.฀ Indirect฀immunoperoxidase฀staining฀was฀carried฀out฀with฀ monoclonal฀ antibody฀ to฀ rubella฀ virus฀ (Chemicon,฀ USA)฀ followed฀ by฀ incubation฀ with฀ peroxidase-labelled฀ goat฀ anti-mouse฀ IgG.฀ Both฀ primary฀ and฀ secondary฀ antibody฀ incubations฀ were฀ carried฀ out฀ at฀ room฀ temperature฀ for฀ one฀hour.฀The฀peroxidase฀substrate฀3,3’฀diaminobenzidine฀ tetrahydrochloride฀(Sigma,฀USA)฀was฀used฀as฀chromogen.฀ After฀counterstaining฀in฀H&E,฀the฀cells฀were฀examined฀in฀a฀ light฀microscope.฀Immunostained฀negative฀controls฀were฀ included฀by฀replacing฀the฀primary฀antibody฀with฀PBS.

Nested฀PCR฀assay

The฀cultures฀inoculated฀with฀samples฀–฀peripheral฀blood฀ lymphocytes,฀urine฀and฀cerebrospinal฀fluid฀–฀were฀individually฀ snap-frozen฀in฀plastic฀vials,฀stored฀at฀-70°C.฀RNA฀was฀extracted฀ from฀the฀entire฀cell฀pellet฀by฀the฀guanidinium฀isothiocyanate฀ method฀and฀submitted฀reverse฀transcriptase-PCR฀(RT-PCR)฀ followed฀by฀Nested฀PCR฀as฀described฀by฀Best(2)_.

Results

The฀ fibrochondrocyte฀ cells฀ maintained฀ their฀ capacity฀ to฀ form฀ chondro-like฀ structures฀in฀ vitro฀ and฀ maintained฀ tissue฀ organization.฀ The฀ cells฀ morphologically฀ consisted฀ of฀elongated฀fibroblast-like฀cells,฀which฀form฀good,฀well-maintained฀monolayers฀(Figure฀1A).

infected฀ with฀ the฀ sample฀ show฀ antigens฀ appearing฀ as฀ a฀ dark-brown฀stain฀in฀the฀cells.฀Close฀to฀the฀nucleus,฀there฀was฀ a฀circular฀shape฀seen฀with฀a฀contraction฀of฀the฀cytoplasm฀ around฀the฀altered฀nucleus฀(Figure฀1C).

Titrations฀were฀done฀using฀tenfold฀dilutions฀of฀the฀virus฀ inoculated฀into฀plate฀cultures/dilutions฀with฀the฀same฀set฀ of฀dilutions฀used฀for฀all฀two฀cell฀types.฀During฀the฀growth฀ of฀the฀virus,฀medium฀DMEM฀only฀was฀used฀to฀maintain฀the฀ cultures฀and฀no฀fluid฀changes฀were฀made.฀The฀infection฀ curve฀increased฀during฀the฀five฀days฀of฀observation,฀and฀ showed฀that฀the฀titers฀in฀the฀fibrochondrocyte฀cells฀were฀ higher฀than฀those฀observed฀in฀the฀Vero฀cell฀line.฀The฀titers฀ were:฀Vero฀cells:฀105,51_{฀(cerebrospinal฀fluid),฀10}5,90฀_(blood),฀

106,05_{฀ (urine)}฀_TCID

50/0.1mL;฀ and฀ fibrochondrocytes฀ cells:฀

106,45_{฀ (cerebrospinal฀ fluid),฀ 10}6,92_{฀ (blood),฀ 10}7,05_{฀ (urine)฀}

TCID₅₀/0.1mL฀(Figures฀2A฀and฀B).

The฀viral฀genome฀was฀extracted฀from฀fibrochondrocytes฀ inoculated฀with฀samples฀and฀RA฀27/3฀strain฀after฀four฀days฀of฀ the฀incubation.฀The฀viral฀genome฀was฀amplified฀by฀Nested฀ PCR฀product฀visualized฀in฀2%฀agarose฀stained฀with฀ethidium฀ bromide,฀and฀had฀an฀expected฀length฀of฀243฀base฀pairs.

All฀stock฀primary฀cultures฀maintained฀in฀our฀laboratory฀ were฀ checked฀ from฀ time฀ to฀ time฀ for฀ the฀ presence฀ of฀ mycoplasma฀and฀other฀contaminants฀and฀the฀results฀were฀ negative.฀The฀preservation฀of฀fibrochondrocyte฀cells฀was฀ possible฀by฀freezing฀in฀liquid฀nitrogen.฀The฀cells฀were฀frozen฀

in฀the฀presence฀of฀10%฀fetal฀calf฀serum฀(FCS)฀plus฀10%฀ DMSO.฀Various฀successful฀recoveries฀from฀liquid฀nitrogen฀ storage฀have฀been฀made฀in฀our฀laboratory฀and฀we฀have฀ obtained฀satisfactory฀results.

Discussion

Rubella฀ virus฀ is฀ the฀ causative฀ agent฀ of฀ the฀ disease฀ commonly฀known฀as฀German฀measles.฀However,฀rubella฀can฀ cause฀complications฀with฀transient฀joint฀involvement฀such฀ as฀arthritis฀and฀arthralgia.฀Interestingly,฀these฀symptoms฀are฀ more฀prevalent฀and฀severe฀in฀RV-infected฀women฀than฀in฀ RV-infected฀men(4,฀15)_{.฀The฀frequency฀with฀which฀rubella฀virus฀}

infects฀joints฀during฀acute฀infection฀and฀the฀percentage฀of฀ these฀joint฀infections฀that฀result฀in฀arthritis฀are฀unknown.

RV฀ infects฀ many฀ culture฀ cell฀ lines฀ but฀ can฀ establish฀ productive฀infection฀only฀in฀a฀limited฀number฀of฀cell฀lines.฀ The฀ cell฀ lines฀most฀ used฀ for฀ virus฀ growth฀ are฀ RK₁₃,฀ SIRC฀ and฀Vero(2,฀20)_.

The฀present฀report฀describes฀the฀growth฀and฀titration฀ of฀the฀rubella฀virus฀isolated฀in฀samples฀of฀fibrochondrocyte฀ cells฀in฀comparison฀the฀Vero฀cell฀line.฀The฀fibrochondrocyte฀ cells฀in฀vitro฀form฀a฀three-dimensional,฀multilayered฀structure฀ that฀resembles฀a฀cartilaginous฀tissue(6,฀1)_.

These฀cells฀infected฀with฀rubella฀virus฀induced฀detectable฀ CPE฀after฀24฀hours฀of฀the฀inoculation฀and฀are฀easily฀visualized฀ in฀ the฀ light฀ microscope฀ and฀ after฀ immunoperoxidase฀ Figure฀1฀–฀Cytopathic฀effect฀of฀rubella฀virus฀in฀fibrochondrocyte.฀A:฀Uninfected;฀

B:฀Cells฀infected฀after฀5฀days฀with฀positive฀rubella฀sample฀(urine).฀Observe฀the฀ cytopathic฀effect฀is฀characterized฀by฀a฀formation฀of฀foci฀consisting฀of฀small,฀circular฀ cells,฀(100x);฀C:฀Immunoperoxidase฀staining฀fibrochondrocyte฀cells฀infected฀for฀3฀ days฀with฀urine฀sample.฀Note฀viral฀antigens฀appear฀as฀a฀dark฀brown฀stain฀in฀the฀ cells.฀Note฀circular฀cells.฀(100x)

A

B

C

Figure฀2฀–฀A:฀Curve฀of฀the฀increased฀infectivity฀of฀fibrochondrocyte฀cells฀infected฀ with฀samples฀(urine,฀blood฀and฀cerebrospinal฀fluid)฀and฀observed฀during฀5฀days;฀฀ B:฀Vero฀cells฀infected฀with฀the฀same฀samples

A

staining.฀Reports฀by฀Chantler฀and฀Cunningham฀described฀

rubella฀ virus฀ in฀ human฀ chondrocyte฀ and฀ synovial฀ cells฀ showing฀detectable฀CPE฀and฀these฀cells฀have฀been฀shown฀ to฀be฀as฀effective฀for฀study฀of฀rubella฀virus(3,฀5)_.

The฀growth฀curve฀for฀the฀rubella฀virus฀in฀fibrochondrocyte฀ cells฀was฀higher฀compared฀with฀the฀Vero฀cell฀line.฀This฀is฀ important฀because฀it฀shows฀that฀cells฀can฀lose฀susceptibility฀ for฀virus฀while฀rubella฀virus฀is฀replicating.

The฀association฀of฀rubella฀virus฀with฀acute,฀transient฀joint฀ manifestations,฀after฀both฀natural฀infection฀and฀vaccination,฀ has฀been฀recognized฀for฀many฀years(4,฀16,฀17)_{,฀but฀the฀search฀}

for฀persistent฀rubella฀virus฀isolation฀in฀subjects฀with฀chronic฀

joint฀symptoms฀following฀rubella฀or฀rubella฀immunization฀ has฀been฀unsuccessful(8,฀17)_.

Recently,฀Lund฀demonstrated฀that฀the฀growth฀of฀rubella฀ virus฀strains฀vary฀in฀their฀abilities฀to฀replicate฀and฀persists฀ in฀cell฀cultures฀derived฀from฀human฀joint฀tissues,฀and฀that฀ this฀arthrotropism฀appears฀to฀be฀linked฀to฀their฀association฀ with฀joint฀symptoms฀in฀vivo(13)_.

These฀findings฀indicate฀that฀fibrochondrocyte฀cells฀could฀ be฀a฀useful฀tool฀to฀investigate฀the฀arthrotropism฀of฀different฀RV฀ isolates.฀The฀fibrochondrocyte฀cultures฀have฀the฀advantage฀of฀ being฀easier฀to฀manipulate฀and฀providing฀greater฀consistency฀ for฀comparative฀experimentation฀than฀organ.

References

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13.฀LUND,฀K.฀D.;฀CHANTLER,฀J.฀K.฀Mapping฀of฀genetic฀determinants฀ of฀rubella฀virus฀associated฀with฀growth฀in฀joint฀tissue.฀Journal฀ of฀Virology,฀v.฀74,฀n.฀2,฀p.฀796-804,฀2000.

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with฀ development฀ of฀ prolonged฀ arthritis,฀ neurological฀ sequelae,฀ and฀ chronic฀ rubella฀ viremia.฀ J฀ Infect฀ Dis,฀ v.฀ 152,฀ p.฀606-12,฀1985.

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Virology.฀New฀York:฀Raven฀Press,฀1990.฀p.฀815-38. 1.฀ARAÚJO,฀V.฀G.฀et฀al.฀In฀vitro฀rapid฀organization฀of฀rabbit฀meniscus฀

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